Abstract

Site-directed mutagenesis of the γ-aminobutyric acid type A (GABA_A) receptor β₂ subunit has demonstrated that conversion of a conserved glycine residue located at the entrance to the first transmembrane domain into the homologous ρ₁residue phenylalanine alters the modulating effects of four different i.v. anesthetics: pentobarbital, alphaxalone, etomidate, and propofol. Using the baculovirus expression system in Spodoptera frugiperda 9 cells, anesthetic-induced enhancement of [³H]muscimol and [³H]flunitrazepam binding in receptors containing the β₂(G219F) point mutation displayed a significantly reduced efficacy in modulation by all four i.v. anesthetics tested. Furthermore, GABA_A receptors containing the α₁(G223F) point mutation also significantly decreased the maximal effect of etomidate- and propofol-induced enhancement of ligand binding. Conversely, the homologous point mutation in ρ₁ receptors (F261G) changed the i.v. anesthetic-insensitive receptor to confer anesthetic modulation of [³H]muscimol binding. Consistent with the binding, functional analysis of pentobarbital-enhanced GABA currents recorded with whole-cell patch clamp demonstrated the β₂(G219F) subunit mutation eliminated the potentiating effect of the anesthetic. Similarly, propofol-enhanced GABA currents were potentiated less in α₁β₂(G219F)γ₂ receptors than in α₁β₂γ₂ receptors. Although ligand binding displayed comparable K_D values for muscimol among wild-type, α₁β₂γ₂, and mutant receptors, patch-clamp recordings showed that α₁β₂(G219F)γ₂ receptors had a significantly more potent response to GABA than did α₁β₂γ₂ or α₁(G223F)β₂γ₂. The α₁β₂(G219F)γ₂ receptors also were more sensitive to direct channel activation by pentobarbital and propofol in the absence of GABA. These results suggest that the first transmembrane glycine residue on the β₂ subunit may be important for conformational or allosteric interactions of channel gating by both GABA and anesthetics.